Identification of cry gene from Bacillus thuringiensis by PCR and isolation of unique insecticidal bacteria

A gene identification method utilizing polymerase chain reaction (PCR) was investigated in order to evaluate its application to Bacillus thuringiensis cry genes. A major objective of this study was to predict the insecticidal spectrum of a newly isolated B. thuringiensis strain based on its gene contents. B. thuringiensis is known for its production of insecticidal proteins. Since these proteins are crystallized in the bacterial cells, they are called crystal proteins and their genes "cry." Numerous cry genes encoding for the insecticidal proteins have been identified. In this study, those genes were cloned and expressed in Escherichia coli to obtain pure proteins individually. Insecticidal activities of these proteins were determined in Bombyx mori, Plutella xylostella, Spodoptera litura, Aedes japonicus, Anomala cuprea. Proteins encoded by seven type-I cry genes, cryIAa, cryIAb, cryIAc, cryIB, cryIC, cryID and cryIE, were found to be active against these lepidopteran species. However, activity level of each protein differed significantly. For example, the CryIAa protein was found to be very active against B. mori but not active against S. litura. The study further indicated that the CryIIA, CryIVA, CryIVB, CryIVC and CryIVD proteins were active against A. japonicus, the mosquito species used in this study, while CryIIB was not. The CryIIIA protein has been reported to be active against coleopteran species such as Leptinotarsa decemlineata, but no activity was found against A. cuprea. In order to determine cry gene types in B. thuringiensis isolates by the PCR method, a set of specific primers were designed for each gene and synthesized by a DNA synthesizer. The study demonstrated that these primers were specific enough to determine cry gene types in B. thuringiensis strains. A number of B. thuringiensis isolates were then screened with this PCR method, and their cry gene types were determined